There are numerous inflammatory skin diseases, which prompt millions of patients to visit dermatology clinics annually. In most cases, cutaneous pathologies are caused by irritant, and even pathogen, agents. Among these diseases, however, there are some where homeostasis of the immune system is compromised; psoriasis, considered a possibly autoimmune disease, and allergic contact dermatitis (ACD) are two examples of this type of pathology.
Psoriasis is a chronic inflammatory skin pathology affecting between 2-3% of the world population. In most clinical cases, 20% of these patients may develop rheumatoid arthritis, complicating their quality of life. The symptomatology of this disease transcends merely esthetic concerns involving the formation of erythematous plaques in the skin, affecting elbow and knee joints, posing complications to the patient's clinical picture. This variant of the disease is usually diagnosed erroneously because it has a similar symptomatology to arthritis.
On the other hand, contact dermatitis is one of the world's most common work-related diseases, as it is caused by prolonged exposure to certain commonly non-immunogenic molecules (hapten). In such cases, constant exposure to the hapten may trigger highly inflammatory hypersensitive events since this molecule can covalently bond to the patient's own proteins. Covalent bonding between the patient's own protein and the hapten generates a new antigen that now does have immunogenic properties inducing a more powerful immune response.
In the case of psoriasis and ACD, even though they are pathologies with different etiologies, they do have the same cellular networks, such as dendritic cells residing in the skin (Langerhans cells, dermal cells), circulating T lymphocytes, macrophages, etc., which collectively induce a circuit of cellular and humoral components that converge in inflammatory processes triggered by immunogenic stimuli.
The skin is the most extensive organ of the human body. In some areas, it is 0.5 mm thick, as in the eyelids, whereas in other parts, as in the palms of the hands or soles of the feet, it reaches a thickness of up to 5 mm. Histologically, the skin is subdivided into three layers: 1) the epidermis, which is the outermost layer; 2) the dermis, or the middle layer; and 3) the hypodermis, which is the deepest layer, basically made up of subcutaneous fatty tissue.
The skin is, along with the mucous epithelium, the first physical defense barrier against the entry of different microorganisms, many of which can be pathogenic. In particular, the epidermis maintains direct contact with the external environment and consists of a stratified epithelium, made up of basal cells involved in tissue regeneration and more differentiated suprabasal cells, known as keratinocytes. As cell differentiation progresses, the keratinocytes express high concentrations of keratin. This protein gives cells the capacity to resist mechanical and chemical abrasion, and in addition provide great impermeability to the epidermis. When keratinocytes reach a high concentration of keratin and lipids in their cytosol, they lose their nuclei and die, forming the stratum corneum.
Keratinocytes can be activated through their cytokine receptors or pathogen recognition receptors (PRR) and secrete a wide range of cytokines to the microenvironment, for example, interleukin 1 (IL-1), IL-6, TNF-α, TGF-β1, IFN-γ, chemokines, such as IL-8, and lectins, such as Galectin (Gal)-1, -3, and -7.
All these molecules secreted by the keratinocytes can modulate cell activity in the resident skin immune system, such as Langerhans Cells (LCs), polymorphonuclear cells (PMNs), and even T cells crossing and migrating through this tissue.
T lymphocytes are key components in the adaptive immune response, which is evident three days after innate immune response mechanisms are activated. At this stage, dendritic cells (DCs) act as a link between the innate immune response and the adaptive immune response. LCs are immature antigen-presenting cells, residing in the epidermis, and like all immature dendritic cells, they have a high endocytic capacity and a low antigen-presenting ability. Nevertheless, upon invasion by a pathogen, resident dendritic cells endocyte the pathogen, maturate, and migrate to draining lymph nodes. This maturation consists in the presentation of endocyte antigens (Ag) within the context of a class II major histocompatibility complex (MHC II), which induces specific activation of virgin T lymphocytes in the secondary lymphatic organs. Activation of the lymphocytes requires three specific signals: 1) one between the antigen/MHC II and T cell receptor (TCR); 2) interaction between CD28 co-receptor (T cell) and CD80/CD86 co-receptor (APC); and, finally, 3) the microenvironment of cytokines secreted by the APC. This last signal is mainly responsible for the type of effector T response. After antigen presentation, lymphocyte T αβ CD4+ can acquire different sub-class of T helper cells: T helper 1 (Th1), Th2, Th9, Th17, Th22, and regulatory T cells.
The Th1 cells are a subtype of T-lineage lymphocytes, which are differentiated from naïve T cells in the presence of IL-12 and IL-18 secreted from the antigen-presenting cell. These lymphocytes are characterized for secreting high levels of IFN-γ, a potent activator of phagocytic/degradative activity in macrophages, known as the “oxidative burst.” This activation determines that macrophages increase the expression of the CD80 and CD86 molecules, MHC I and II molecules, CD40, chemokines (IL-8 and MCP-1), inflammatory cytokines (IL-1 and TNF-α), and oxygen reactive species (superoxide anion, nitrogen oxide [NO], etc.).
In particular, participation of Th1 lymphocytes has been described in chronic inflammatory processes, whereas Th17 lymphocytes are the cells that maintain the inflammatory foci during the chronic stage of the pathology. The Th17 cells are characterized for secreting IL-17 (present as A or F isoforms), which acts as a chemokine of neutrophils, and IL-22. This T response stimulates neutrophilia (by secretion of G-CSF) and neutrophil recruitment at the infection site. It also stimulates macrophages to produce proinflammatory cytokines, stimulates TNF-α secretion, induces metalloproteinase secretion, and promotes the production of mucus as well as antimicrobial peptides, chemokines, and IL-6. This last interleukin, along with TGF-β1, promote cell differentiation to Th17 profile in T cells activated by IL-2. In addition, IL-23 makes the activated Th17 expand and produce more IL-17 and IL-6. In this regard, a T cell subtype, the Th22 lymphocyte, specific producer of IL-22, has been described recently promoting proliferation of keratinocytes. This cytokine is mainly responsible for the formation of erythematous plaques characteristic of psoriasis.
Lastly, regulatory T cells (Tregs) are a subtype of lymphocytes of T αβ lineage particularly focused on negatively regulating immune response. There are two types of Tregs, natural Tregs (nTregs) and inducible Tregs (iTregs) (Sakaguchi et al., 2005). The nTregs mature in the thymus and present markers characteristic of CD4, CD25high and the Foxp3 transcription factor. On the other hand, iTregs are differentiated in secondary lymphoid organs from naïve CD4+ CD25− T cells in the presence of TGF-β1 and are also CD4+ CD25highFoxp3+. Moreover, a subpopulation of iTRegs known as Tr1 has been described. Tr1 is differentially generated in the presence of IL-27, does not express the Foxp3 transcription factor (Foxp3−) (Roncarolo et al., 2001, Ilarregui et al., 2009) but selectively expresses the LAG-3 and CD49d markers (Gagliani et al., 2013). Numerous studies based on selectively eliminating this population or blocking their activity suggest that Tregs basically participate in promoting homeostasis of the immune response (Valencia and Lipsky, 2007; Vignali et al., 2008).
Tregs express CTLA-4, FAS-L or secrete IL-10, IL-35, and TGF-β1, that can induce cell death or anergy of effector T cells, contributing to the resolution of the immune response and to the maintenance of peripheral tolerance (Curotto de Lafaille and Lafaille, 2009). Thus, differentiation of naïve T cells to a regulatory T lineage in patients with autoimmune diseases such as psoriasis could have the capacity of resolving the inflammatory process. Accordingly, stimulation of the skin's immune cells with molecules that positively modulate the differentiation of T naïve to regulatory T cells can be an attractive treatment option for chronic skin pathologies.
In psoriasis pathologies, keratinocytes have an unregulated mechanism of proliferation, apoptosis, and differentiation promoting the production of erythematous plaques, acanthosis (increase in epidermal thickness) and parakeratosis (an incomplete keratinization characterized by the retention of nuclei in the stratum corneum) (Baadsgaard et al., 1990). Furthermore, the initial cause that triggers the pathology and the circuits leading to their resolution are unknown (Abrams et al., 2000; Goedkoop et al., 2004; Lew et al., 2004). The general consensus on this autoimmune disease states that there are genetic factors associated to a possible initial infection that contribute to triggering this chronic inflammatory skin response.
One such consequence of the parakeratosis process is a reduction in Gal-7 expression (a lectin preferentially expressed in keratinocytes) in the deepest layers of the epidermis (Magnaldo et al., 1995), as a symbol of a deficiency in the epithelial stratification. This deficiency in epidermal differentiation is accompanied by an important compromise in the inflammatory infiltrate of Th1 and Th17 lymphocytes, macrophages, and DCs. In the acute stage, Th1 lymphocytes are the first recruited in the skin, and by secreting IFN-γ they contribute to the inflammatory environment characteristic of the first stage of the pathology. In the subsequent chronic stage, however, the Th1 lymphocyte infiltrate diminishes, and Th17 cells overcome in this second stage. The Th17 lymphocytes located in the affected tissue secrete high concentrations of IL-17, -21, and -22, although the greater source for the production of IL-22 is represented by the recently identified Th22 lymphocyte subpopulation. There is a difference between the mouse model and humans. The presence of Th22 is largely documented in patients with psoriasis, but it was not possible to characterize this T lymphocyte subpopulation in mice. In animals, Th17 lymphocytes contribute as the main source of IL-22 (Awasthi et al., 2009; Ciric et al., 2009; Sutton et al., 2009).
Recently, blocking anti-IL-21 antibodies were observed to reduce inflammation, infiltration of the immune system cells, and proliferation of keratinocytes in experimental murine models that had received xenotransplants from psoriatic patients (Caruso et al., 2009). At the same time, during the course of the disease, an increased expression of the p53 protein was observed in patient lesions (Baran et al., 2005). Interestingly, Gal-7 is a lectin that was described and identified as a p53-regulated protein (Kopitz et al., 2003).
In addition, TNF-α, a proinflammatory cytokine involved in the activation and migration of LCs, is an essential factor in the development of this pathology (Marble et al., 2007). Several clinical trials have shown that TNF-α blocking produces a significant improvement in patients with psoriasis. The underlying mechanism of this therapeutic effect is still under controversy, but recent studies have revealed that treatment with ETANERCEPT, a soluble TNF-α-blocking receptor, produces a rapid decrease in inflammation and an increase in the apoptosis of dermal dendritic cells (DCs).
Contact Hypersensitivity (CHS) is an inflammatory skin model that is widely used in studies on inflammatory skin diseases because the induced immunological mechanisms are similar to the ones manifest in human allergic contact dermatitis (ACD). In short, ACD has a number of well-established phases, the first of which is the initiation or sensitization phase. Haptens are low molecular weight molecules that have no immunogenic capacity and act as a sensitizing molecule during this phase. Moreover, haptens can undergo modifications in the skin, such as covalent bonding to the patient's proteins, thus acquiring immunogenic capacity (called carrier proteins).
At the initial phase of sensitization, the DCs, now activated by the new antigen (hapten-carrier), mature and migrate to the lymph nodes, presenting the antigen to T cells. The activated T lymphocytes proliferate and migrate from the lymph nodes, remaining in circulation until they enter into contact with the hapten-carrier within the context of the MHC II in an antigen-presenting cell.
The second phase or inflammatory phase is triggered by a subsequent stimulus with the allergen and it can be divided in two stages: an early stage (2-hour post re-stimulation with the allergen) and a late stage (24 hours after re-stimulation), each one of them is characterized by a specific cellular and humoral profile. Finally, 48 hours post-contact with the allergen, the inflammation decreases due to the activity of the regulatory T cells (Tregs), CD4+, CD25+, FoxP3+, as well as IL-10-secreting Tr1 cells (Allan et al., 2008). The regulatory capacity of these cells is manifest both at the level of the draining lymph nodes as well as at the periphery of the skin, where they inhibit clonal expansion of T CD8+ lymphocytes (Fas-FasL pathway and the interaction between CTLA-4 and CD80/CD86) (Tan et al., 2014).
By contrast, Irritant Contact Dermatitis (ICD) has no first sensitization phase as in CHS. Recent studies show that LCs migrates from the skin to the lymph nodes after topical application of an irritant. At the same time, the infiltrate responds (resident CD4+ and CD8+) to stress and chemokines secreted by the keratinocytes of the epidermis, as well as dermal fibroblasts. These fibroblasts are exposed to the irritant because epidermal irritation alters cutaneous permeability, enabling the inflammatory agent to reach the dermis. Thus, both ACD as well as ICD share in great measure the cellular and humoral components present in the immune response during the inflammatory process.
Furthermore, the presence of inflammatory cytokines on Th1 and Th17 cells has been observed during cutaneous irritation; they are also present in the skin of psoriatic patients. Therefore, the use of both experimental models might allow for elucidating the immunological circuits operating during the skin's inflammatory response.
Even though both inflammatory pathologies are considerably different as regards the factor that triggers immune response, the type of infiltrate that characterizes them as well as the duration and magnitude of the inflammatory process have something in common: the DCs that capture and present antigens to T cells are intimately tied to the onset of the pathologies.
Recent studies, however, indicate that skin DCs can act dependent on the microenvironment inducing T lymphocytes to either inflammatory profiles (Th1, Th17, etc.) or tolerogenic or anti-inflammatory profiles (iTreg, Tr1).
Langerhans Cells (LCs) are professional antigen-presenting cells (CPA) located in the basal and suprabasal regions of the epidermis, where they interact continuously with the keratinocytes. The LCs come from bone marrow, have a characteristic CD1+CD34+Langerin+ phenotype, and are bound to the keratinocytes by E-cadherin-mediated bonds, forming a network of antigen-presenting cells in the epidermis.
LCs are not the only Langerin+/− antigen-presenting cells of the skin. In addition, there are Langerin+ dendritic cells (DCs) and Langerin− dermal DCs in the dermis, complicating the scenario even more in the attempt to identify who triggers the immune response with a specific stimulus and who regulates negatively the response. Pioneer work sustained that all skin DCs were responsible for capturing foreign antigens, then migrating to lymph nodes and presenting the antigens to T lymphocytes (LT) in order to trigger an adequate immune response (Hemmi et al., 2001). In the past decade, however, this premise was found not to be as conclusive as originally proposed; instead, it is now known that there is a complex mechanism where each DC subpopulation in the skin may play a specific role.
In turn, the same DC population is capable of participating in either inflammatory or anti-inflammatory, dependent on the prevailing cytokines in the microenvironment during the activation process. This microenvironment is determined mainly by the cytokines and activation of the LCs can modify its physiology and the glycosylation pattern (or glycophenotype) of the membrane proteins. Various studies have shown that differential glycosylation of proteins plays a fundamental role in the functioning and homeostasis of the immune system (Demetriou et al., 2001) as it affects interactions between cells, and between cells and proteins present in the extracellular matrix. Indeed, the glycome of a cell can modify the specific bond of membrane glycoproteins to proteins present in the extracellular environment; activate signaling cascades; or retain receptors in the membrane, thus modifying the type of response of such cells.
The enzymes that synthesize glycan structure and the remodeling of saccharide that constitute the glycoproteins are called glycosyltransferases and glycosidases (Marth and Grewal, 2008). These enzymes are part of the rough endoplasmic reticulum and the Golgi apparatus, where saccharides are incorporated and eliminated sequentially. Moreover, this biosynthesis process is finely regulated and coordinated by chaperone proteins that, in conjunction with the glycosyltransferases and glycosidases, synthesize the final glycoprotein structure (Rabinovich and Toscano, 2009; Van Kooyk and Rabinovich, 2008). These glycans play essential roles in cell physiology, as they are involved in cell adhesion, migration, subcellular traffic, endocytosis, signal transduction, receptor activation, etc.
There are several checkpoints involved in the biosynthesis of specific glycoproteins. Nonetheless, the main mechanism for switching the glycophenotype consists in recycling protein by endocytosis and the subsequent synthesis of new molecules subject to the differential activity of glycosyltransferases and glycosidases regulated by different cellular stimuli.
Information codified in the glycome is decoded by different protein families called lectins or glycan-binding proteins. These molecules have a high affinity to different saccharide residues of cell surface glycoproteins. These lectins can also be divided into different groups according to their evolutionary structural relationship and their affinity to carbohydrates: 1) Siglecs (associated to the cell surface), 2) C-type Lectins (associated to the cell surface), and 3) Galectins (soluble molecules for intracellular and extracellular localization).
As mentioned before, the responsibility for decoding the biological information contained in the glycome lies, at least in part, in a group of proteins known as galectins (Leffler et al., 2004). These proteins present a preferential affinity to repeat units [Galβ1-4-NAcGlc] in both N- as well as O-glycans of the glycoprotein cell surface and extracellular matrix (Salatino et al., 2008). This bonding to glycosidic residues occurs through a carbohydrate recognition domain (CRD, approximately 130 amino acids) that is highly conserved in all its galectins (Cooper et al., 2002). In addition, depending on its biochemical structure, galectins are classified in three groups: “prototype galectins” (Gal-1, 2, 5, 7, 10, 11, 13, 14, y 15), “chimeric galectin” (Gal-3), and “tandem-repeat galectin” (Gal-4, 6, 8, 9, y 12) (Liu and Rabinovich 2005; Yang et al., 2008).
Galectins can be expressed in all animal species, and many of them are tissues and compartments specific. Recently, diverse intracellular and extracellular functions have been described (Rabinovich et al., 2007; Rabinovich and Toscano, 2009).
Even though these proteins are secreted by the cells and are mostly located in the extracellular environment, there is no peptide signal in their amino acid sequence. Consequently, their secretion is independent of the classic ER and Golgi apparatus pathways; on the other hand, these proteins are secreted by an a typical mechanism known as ectocytosis (Yang et al., 2008).
In recent years, this lectin family has been linked to various biological processes as regulators of homeostasis in the immune response (Rabinovich and Toscano, 2009), tumor progression (Liu and Rabinovich, 2005), and neovascularization (Cardenas Delgado et al., 2010; Markowska et al., 2010; Thijssen et al., 2006; Croci et al., 2014). Some galectins, such as Gal-1 and Gal-3, are expressed in a broad range of tissues, whereas other galectins have a more restricted expression pattern, such as Gal-4 in the gastrointestinal system, Gal-10 in eosinophils, Gal-12 in adipose tissue, and Gal-7 in keratinocytes.
Both Gal-7 as well as Gal-1 are expressed in keratinocytes and share their affinity to LacNAc repeats. However, affinity of Gal-1 to LacNAc terminal units is greater than Gal-7. Another important difference between these two proto-type galectins is that Gal-1 binding is sterically prevented when the oligosaccharide has sialic acid residue in the α2,6 terminal position (this is not so in the α2,3 sialylation position). On the other hand, neither of these two positions of the sialic acid residue modifies Gal-7 binding. In this sense, the differential binding capacity of these two lectins expressed by keratinocytes can induce different signaling pathways.
Gal-7, an endogenous lectin preferentially expressed on keratinocytes (Saussez and Kiss, 2006), was discovered simultaneously in two laboratories: one was studying genes that respond to retinoic acid (Madsen et al., 1995) and the other was looking for genes inducible by oncogen p53. Gal-7 is expressed in stratified epithelia and it has been claimed that Gal-7 might contribute to tissue homeostasis (Gendronneau et al., 2008). Nonetheless, it has been observed that this lectin is also expressed in the trachea and the ovary, two unstratified epithelia (Sao et al., 2002). This lectin is distributed in the cell-to-cell contact regions, particularly in the outermost layers of the epidermis, as well as in the esophagus epithelium, the oral cavity, the cornea, and Hassall's corpuscles in the thymus of mice, rats, and humans.
Even though the role of Gal-7 in tissue immunity has not been completely elucidated, various functions of this lectin regarding epithelial homeostasis have been described, under physiological as well as pathological conditions.
Cell Migration:
Using an in vivo model consisting of cornea lesions induced by UV radiation, topical treatment with recombinant Gal-3 (rGal-3) and recombinant Gal-7 (rGal-7) induced greater scarring of the lesions in relation to the different growth factors (Cao et al., 2002; Cao et al., 2003).
Apoptosis Regulation:
Given that one of the molecular mechanisms induced by Gal-1 binding to its specific ligands is the activation of the apoptosis pathway, the function of Gal-1, Gal-2, Gal-9, and Gal-7 as pro-apoptotic effectors has been studied in different cell models. In particular, in colon cancer DLD-1 cells, the p53-dependent apoptosis pathway evidenced an increase in Gal-7 expression, among other 7002 genes. In other related work, UVB radiation was shown to induce p53 expression on keratinocytes, leading to an increase in Gal-7 levels (Bernerd et al., 1999).
Effect on Tumor Progression:
The role of Gal-7, like in the majority of galectins, in tumor progression/development, is controversial. In certain tumor cell lines, it has anti-tumor effects, whereas in others, it has pro-tumor functions. Both in vivo and in vitro models have shown that DLD-1 tumor cells transfected with Gal-7 have a lower tumor growth rate independently of cell apoptosis (Ueda et al., 2003). This same anti-tumor property of Gal-7 has been observed in human neuroblastoma cells (Kopitz et al., 2003).
Tissue Differentiation Marker:
It has been claimed that Gal-7 may be used as a tissue differentiation marker given that, at the onset of its expression on keratinocytes, it coincides with the beginning of epidermal stratification (Magnaldo et al., 1995; Magnaldo et al., 1998; Timmons et al., 1999) and its level of expression is higher in areas of the skin where the epidermis is made up of a greater number of suprabasal cell layers.